In order to test the hypothesis programmed cell death in lymphocytes involves activation of an endogenous protease, we have tested the ability of several cysteine protease inhibitors to block the TcR-induced death of the CD4+ T cell hybridoma 2B4. Two chemically distinct families of inhibitors of calpains and cathepsins, the E-64 and leupeptin families, were found to rescue 2B4 cells from death measured by trypan blue or propidium iodide uptake after overnight culture on immobilized alpha-CD3 or alpha-TcR antibodies. DNA breakdown measured by flow microfluorimetry was also blocked. These drugs enhanced the TcR-induced IL-2 secretion by as much as 9-fold, showing that they do not act by interfering with signal transduction. Other programmed cell death systems in lymphocytes tested were the steroid-induced death of CD4+CD8+ thymocytes, which was blocked by leupeptin family inhibitors, and the CH31 B cell line, whose mIgM-induced death was unaffected by these inhibitors. We have also found that short-term-cultured lymphocytes can undergo programmed cell death after TcR cross-linking. After activation of purified resting mouse lymph node T cells (or PNA+ thymocytes) by immobilized alpha-CD3 or alpha-TcR antibodies, continuously dividing cells were be maintained in IL2 for periods of days to weeks. When re-exposed to surface-bound antibodies against TcR or CD3, these cells were found to undergo a decrease in cell division and mitochondrial metabolism, accompanied by substantial cell death. When purified populations of CD4+ and CD8+ cells were cultured this way, the cell death effect was principally seen in the CD4 population, with more modest effects in all responses in the CD8 population. In the CD4+ cells DNA breakdown was clearly seen to accompany cell death when analyzed by flow microfluorimetry. This effect was blocked by cyclosporin, co-culture with splenic macrophages, or cysteine protease inhibitors.